Western blot kit for detection of vaccinated poultry

a technology for poultry and western blot, which is applied in the field of western blot kit for poultry detection, can solve the problems of difficult detection of avian influenza, trade restrictions, and inability to distinguish between vaccinated birds and naturally infected birds using commonly available diagnostic tests

Inactive Publication Date: 2013-01-17
NANO JAV DARU
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

One limitation of killed whole-virus vaccines is that vaccinated birds cannot be distinguished serologically from naturally infected birds using commonly available diagnostic tests.
Thus detection of avian influenza becomes much more difficult and often results in trade restrictions because of the inability in DIVA strategy to distinguish betwe

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0017]To develop the western blot kit, four kinds of poultry serum provided, first was influenza infected serum collected from infected chickens in the field, second was vaccinated serum collected from chickens which are given influenza vaccine, third was experimental infected serum collected from chickens which are infected with H9N2 antigen and the fourth was normal serum collected from healthy chickens.

example 2

[0018]Peptide sequence synthesized by Metabion Co. (Germany), antigens (peptides) with 150 ng / μl concentration diluted in sample buffer and run in to the Tricine-SDS-PAGE gel with a size marker, A protein size marker purchased from sigma, Cat NO: M3546 Ultra low range molecular weight marker (M.W. 1060-26600). 16.6% Urea Tricine-SDS-PAGE made by the protocol which is published by Hermann Schagger in Nature Protocols 1, 16-22(2006). Electrophoresis was conducted at 40 v at room temperature and after each 30 min increase the voltage 10 v till 80 v, until the dye front was near the bottom of the gel.

example 3

[0019]To prepare the Tricine SDS PAGE gel in example 2, the following procedure must be done:

GEL BUFFER PREPARATION:

[0020]

Anode bufferCathode bufferGel buffer(10X)(10X)(3X)Tris (M)1.01.03.0Tricine (M)—1.0—HCL (M) 0.225—1.0SDS (%)—1.00.3pH8.9 8.25 8.45

[0021]16% / 6M Urea Mini Gel:

16% / 6M Urea4% stacking gel(resolving gel)AB-3ml0.5—AB-6ml—5Gel buffer (3X)ml1.55Glycerolg——Ureag—5.4Add water to final volume615Polymerized by adding:APS (10%)μl4550TEMEDμl55AB-3:, 49.5% acrylamide., 3% bisacrylamide.AB-6: 49.5% acrylamide, %6 bisacrylamide,

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PUM

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Abstract

Modified western blot membranes with silver nanoparticle allow the small peptides of the NS1 protein of the poultry influenza virus to be kept in the membrane and not to diffuse during transferring from the Tricine SDS PAGE. These peptides may differentiate infected from vaccinated poultry.

Description

FIELD OF THE INVENTION[0001]This kit could differentiate infected poultry from vaccinated during influenza outbreak.BACKGROUND OF THE INVENTION[0002]Differentiating infected from vaccinated animals is known as DIVA strategy. Vaccination is primarily performed using killed whole virus-adjuvanted vaccines. Proper vaccination may reduce or prevent clinical signs, reduce virus shedding in infected birds, and increase the resistance to infection.[0003]One limitation of killed whole-virus vaccines is that vaccinated birds cannot be distinguished serologically from naturally infected birds using commonly available diagnostic tests. Thus detection of avian influenza becomes much more difficult and often results in trade restrictions because of the inability in DIVA strategy to distinguish between vaccinated and infected individuals.[0004]The most common method to distinguish vaccinated individuals from infected is the use of unvaccinated sentinels. A second approach is the use of subunit va...

Claims

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Application Information

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IPC IPC(8): C12Q1/70G01N21/76B82Y15/00
CPCG01N33/54346G01N33/553G01N2469/20G01N2333/11G01N33/56983
Inventor MADANI, RASOOLREZAYAT, SEYED MAHDISARKAR, SAEEDEMAMI, TARA
Owner NANO JAV DARU
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